Laboratory of Molecular Biophysics
Laboratory Journal 2002
J.M. McDonnell

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J.M. McDonnell

Molecular structures and interactions in immune responses and apoptosis

Overview:

Research in my group focuses on two protein interaction networks: the IgE network, a set of proteins that control allergic and inflammatory immune responses, and the BCL-2 network, a family of related proteins which regulate programmed cell death. Both of these networks control processes critical to human health. Like many biological processes, both immune regulation and the control of apoptosis rely on complex interaction pathways to regulate their activities. In many cases individual molecules have multiple ligands and it is often difficult to know which interaction, or set of interactions, permit or prevent a biological process to occur. Our goal is to define the set of molecular interactions for a given network, describe the structures of the individual molecules or complexes, understand the physical basis of how these interactions occur and then use this information to derive specific inhibitors of interactions. These inhibitors are then be used in in vitro and in vivo assays as probes to dissect complex biological signalling networks to better understand how the process occurs and to define possible ways of controlling these processes.

1. Methods for studying molecular recognition events

Sarah Bagby, Sara Fanning, Giles Robertson, Peter Teriete
Collaborators: Peter Hore (Department of Chemistry, University of Oxford), Martin Noble

Our long term goal is to understand the complete energy landscape of macromolecular recognition events. Macromolecular interactions are very complex; to fully understand how this occurs mechanistically would require a complete description of molecular structure and dynamics for both the receptor and ligand, and an understanding of the energetics and thermodynamics of the interaction, in a fully time-resolved manner throughout the lifetime of the bound complex as well as during the processes of association and dissociation. Towards this end we are employing high-resolution structural methods (X-ray crystallography, NMR spectroscopy) to study molecular structures and dynamics in free and bound states, coupled with methods that allow insights into the dynamics processes of association and dissociation (SPR, fluorescence, NMR spectroscopy, photo-CIDNP, mass spectrometry) [1]. We are developing new methods to describe interaction pathways, using methods to selectively label targets and then follow interactions at atomic resolution through time-resolved NMR techniques. We believe understanding these interactions pathways will allow an unprecedented understanding of the mechanism of molecular recognition and offer new insights to the development of inhibitors of macromolecular interactions.


Figure 1: An SPR sensorgram showing the complex kinetics of the interaction between the Fyn SH3 domain and a polyproline peptide ligand. ...more	Figure 2: Atomic resolution kinetic analysis of molecular interactions using time-resolved photo-CIDNP NMR. ...more

2. Structures and interactions of IgE network components

Lorraine Hewitt, Rick Hibbert, Naomi Price, Peter Teriete
Collaborators: Hannah Gould and Brian Sutton (King's College London), Kurt Drickamer and Pauline Rudd (Glyocbiology Institute, University of Oxford)

2.1. Interaction analysis of IgE binding to its high affinity receptor FceRIa

Interactions between immunoglobulin E (IgE) and its receptors are crucially involved at various stages of the allergic response, including the presentation of allergens to the immune system, the regulation of IgE synthesis and perhaps also the switch to IgE production by B cells, and the triggering of mast cells and basophils in the immediate hypersensitivity reaction [2]. We have embarked upon a programme of structural studies of IgE and its high affinity receptor Fc

by NMR spectroscopy, X-ray crystallography and other biophysical techniques. Understanding the molecular details of these interactions, all of which are potential points of therapeutic intervention, is essential for the design and/or screening for inhibitors. Our long time collaborators in this area, Professors Gould and Sutton at King's College London, have recently determined the crystal structure of the complete IgE Fc (Figure 3) [3], and our group has solved the structure of the C

2 domain of IgE by NMR and demonstrated that C

2 binds to Fc

RI (Figure 4) [4].


Figure 3: The structure of IgE Fc, solved by Wan et al. [3] ...more	Figure 4: The NMR structure of IgE Ce2, docked onto the complex of IgE Ce3-4/FceRIa. ...more

2.2. Structure and ligand interactions studies of CD23

CD23 has been shown to regulate a complex network of allergic and inflammatory signalling mechanisms [5]. It has been implicated in the pathogenesis of several human immunological disorders, including allergy, asthma, graft-versus-host disease and rheumatoid arthritis. CD23 functions through interactions with a number of ligands. The binding of different forms of CD23 to different ligands results in a complex, often seemingly contradictory, role for CD23 in immune regulation. We are studying the structure of CD23 and its interactions with IgE, CD21, various carbohydrate ligands, as well as CD23 oligomerization. We are using the structural and interaction studies to drive the development of inhibitors of some of these interactions, with the goal of developing new investigative agents that can help elucidate how these molecular interactions regulate CD23's diverse biological activities in vivo.

3. Molecular interactions of BCL-2 family proteins

Nieshia Williams
Collaborators: Richard Youle (NIH, USA), David Cowburn (New York Structural Biology Center, USA)

Figure 5: The network of interactions made by the CD23 molecule control its complex biology. ...more

Programmed cell death plays a critical role in development and homeostasis in multicellular organisms. These apoptotic pathways are intricately regulated. Modification of these pathways can lead to unregulated cell growth and differentiation; examples of this can be seen in cancer, autoimmunity and neurodegenerative disorders. Programmed cell death plays a critical role in development and homeostasis in multicellular organisms. These apoptotic pathways are intricately regulated. Modification of these pathways can lead to unregulated cell growth and differentiation; examples of this can be seen in cancer, autoimmunity and neurodegenerative disorders. Members of the BCL-2 family of proteins play a critical role in deciding whether a cell will respond to apoptotic signals. Members of this family include both pro- and anti-apoptotic molecules. A frequent characteristic of the family is the ability to form homodimers as well as heterodimers with other members of the family, and part of their apoptotic regulatory activity is mediated by this cross neutralization activity. We are characterizing the structures of several members of this family and attempting to define the interactions that control their activity.

References:

1. J.M. McDonnell (2001) Surface plasmon resonance: insights into the mechanisms of biological molecular recognition. Curr. Opin. Chem. Biol. 5:572-577.
2. H.J. Gould, B.J. Sutton, A.J. Beavil, R.L. Beavil, N. McCloskey, H.A. Coker, D. Fear, L. Smurthwaite (2003) The Biology of IgE and the Basis of Allergic Disease. Ann. Rev. Immunology.
3. T. Wan, R.L. Beavil, S.M. Fabianne, A.J. Beavil, M.K. Sohi, M. Keown, R.J. Young, A.J. Henry. R.J. Owens, H.J. Gould and B.J. Sutton (2002) The crystal structure of IgE Fc reveals an asymmetrically bent conformation. Nature Immunology 3:681-686.
4. J.M. McDonnell, R. Calvert, R.L. Beavil, A.J. Beavil, B.J. Sutton, H.J. Gould, and D. Cowburn (2001) The structure of the IgE Ce2 domain and its role in stabilizing the complex with its high-affinity receptor FceRI. Nature Struct. Biol. 8:437-441.
5. S. Kijimoto-Ochiai (2002) CD23 as a C-type lectin: a multidomain and multifunctional molecule. Cell. Mol. Life Sci. 59:648-664.